Postoperative ileus (POI) is one of the main complication associated with abdominal surgery (AS) procedures. A number of Veterans deployed in the war zones undergo injuries requiring AS. After AS, patients usually develop nausea, vomiting, bloating, and abdominal pain which are major contributing factors to postoperative discomfort. The incidence of ileus is highest in gastrointestinal (GI) surgery with 24% of patients developing ileus and can be as high as 40% in laparotomy patients. The health burden and the cost of prolonged hospitalization due to POI have been estimated to be as much as $1.47 billion annually in the USA, illustrating its large socioeconomic impact. The lack of effective treatments has prompted novel experimental studies to elucidate the underlying mechanisms. Recent insight in the pathophysiology of POI induced by AS have identified intestinal inflammation triggered by handling of the intestine as a contributing mechanism which is clinically a relevant target for treatment. In other inflammatory conditions, there is evidence that resident macrophages within the GI muscularis contribute to both the initiation and the resolution of inflammation through activations of M1 and M2 phenotypes secreting pro- and anti-inflammatory cytokines respectively. Recent studies point to the vagus nerve controlling a cholinergic anti-inflammatory pathway. We previously established that thyrotropin-releasing hormone (TRH) in the brainstem plays a physiological role (including in the cephalic phase) to stimulate the vagus innervating the GI tract. In the last granting period, we reported that intracisternal (ic) injection of TRH prevents the neurogenic (early phase) of POI occurring within 2-h of AS. Our preliminary data obtained at 6-h post-surgery indicate that 1) AS increases M1 but not M2 macrophages and the infiltration of neutrophils in the gastric muscularis externa along with delay gastric emptying (GE); 2) central vagal activation by ic injection of the stable TRH agonist, RX77368 prevents the above increases and reduces the delayed GE induced by AS without modifying basal GE in sham group. In the last granting period, we also established that AS induces a sharp reduction of plasma levels of the prokinetic hormone, ghrelin known to influence vagal activity and the response is prevented by ic TRH before AS. In addition, we obtained preliminary data showing that the novel long acting and brain penetrant ghrelin agonist, HM01 administered orally activates vagal preganglionic motor neurons in the brainstem and prevents AS-induced delayed GE. Based on these reports and exciting supportive preliminary data, we will test 3 HYPOTHESES: Aim 1. AS induces inflammation in the rat gastric muscularis externa through changes in the activation status of M1 or M2 macrophages in the rat gastric muscularis externa. 2. Central vagal stimulation prevents AS-induced delayed GE by activating cholinergic anti-inflammatory pathway with the deactivation of M1 and or the activation of M2 macrophage leading to inhibiting the inflammation in the gastric muscularis externa. 3. The ghrelin agonist, HM01 is a promising candidate via oral administration to reverse POI by its dual potent prokinetic and anti- inflammatory actions through activation of vagal cholinergic pathway. These aims will be achieved in the rat model of AS-induced POI combined with state-of-the art technologies in neuroanatomy (CLARITY technique combined with targeted double or triple labeling, including anterograde tracing and 3D imaging of vagal fibers, enteric neurons and macrophages), molecular biology (Laser microdissection combined with RT-PCR, RNAscope, RT-qPCR, microRNA targeting, MILLIPLEX Multiplex Assays using Bio-RAD Bio-Plex 3D suspension system powered by Luminex xMAP Technology) and functional study (gut motility and chemical stimulation of vagal activity). The completion of these specific aims will make a conceptual advance to target muscularis macrophages by deactivation of M1 and/or activation of M2 as a potential anti-inflammatory strategy and provide the first preclinical data to validate HM01 as a candidate for new therapy for POI.